Supplementary Figure S4 from Aberrant Upregulation of RUNX3 Activates Developmental Genes to Drive Metastasis in Gastric Cancer

Chromatin status relative to RUNX3 peaks at the WNT5A promoter Enlarged image of the ChIPseq analysis of WNT5A locus in Fig. 4.</p

FIGURE 2 from Aberrant Upregulation of RUNX3 Activates Developmental Genes to Drive Metastasis in Gastric Cancer

RUNX3 drives metastasis in gastric cancer cells. A, Subcutaneous xenograft tumors were obtained by inoculation of 1 × 106 HGC-27 control and RUNX3 KO cells, respectively. Typical images are shown (n = 5). B, The weight of tumors was quantified; ****, P t test. C, Liver metastasis model by splenic inoculation of 1 × 106 HGC-27 cells. Representative images of tumors in liver metastasis in control, RUNX3 KO, and RUNX3 KO rescued with reintroduction of RUNX3 (RUNX3 OE) cells are shown (n = 5, respectively). D, Percentage of the metastatic tumor area in the liver tissue was measured using Image J and graphed (mean + SD); **, P P t test. E, Representative images of tumor formation in spleen by inoculation of 1 × 106 HGC-27 cells in control and RUNX3 KO are shown (n = 5, respectively). F, The weight of tumors in spleen was quantified. G, Kaplan–Meier plots in liver metastasis models present overall survival for mice after inoculation of control and RUNX3 KO of HGC-27 cells; *, P t test. H, Orthotopic transplantation model of 1 × 106 HGC-27 cells. Representative images of tumors in stomach (1 in blue, circled by dot line), peritoneal invasion (2, circled by dot line), and liver metastasis (3, indicated by an arrow head) in control, and also tumors in stomach (circled by dot line) in RUNX3 KO cells are shown (n = 5).</p

Supplementary Figure S1 from Aberrant Upregulation of RUNX3 Activates Developmental Genes to Drive Metastasis in Gastric Cancer

Higher RUNX3 expression in gastric cancer patients is associated with advanced stage and disease progression A, B, TCGA gastric cancer patient dataset visualized by XENA software (UCSC, n=591). Comparison of the mRNA expression of RUNX3 between normal tissues (n=38) and primary tumors (n=411) (A) as well as between stage Ia (n=17) and stage IV (n=49) (B) are shown; **, P < 0.01 and *, P < 0.05 by a two-tailed Student t test. C, TCGA gastric cancer patient dataset from microarray (cBioPortal) was obtained to determine RUNX3 mRNA expression in patients with disease-free (n=22) or with recurrence and disease progression (n=5); *, P < 0.05 by a two-tailed Student t test. D, comparison of survival data in RUNX3 high (red, n=349) or low (black, n=526) patients samples using microarrays obtained from KM-plotter database; P < 0.001.</p

FIGURE 6 from Aberrant Upregulation of RUNX3 Activates Developmental Genes to Drive Metastasis in Gastric Cancer

Direct WNT5A activation by RUNX3 would be a therapeutic strategy in gastric cancer. A and B, Representative imaging of immunofluorescence study in gastric cancer specimens (n = 35). A case showing high expression for RUNX3 (red) and WNT5A (green) in Ecadherin positive (blue) cancer cells (A) and a case showing negative RUNX3 and WNT5A expression in E-cadherin positive cancer cells (B) are shown. Arrows indicate the absence of RUNX3 and WNT5A in E-cadherin regions. Dotted boxes indicate enlarged regions. Scale bar: 50 µm. C, Positive correlation between RUNX3 and WNT5A expression in E-cadherin positive cancer cells by immunofluorescence study is indicated statistically (n = 35, Pearson; r = 0.4744, P r = 0.3622, P < 0.05).</p

FIGURE 3 from Aberrant Upregulation of RUNX3 Activates Developmental Genes to Drive Metastasis in Gastric Cancer

RUNX3 drives a metastatic transcriptional program. A, A volcano plot obtained from RNA-seq. A total of 1,005 genes significantly downregulated (P P B, Analysis of enriched pathway obtained from MSIgDB Hallmark 2020 and Gene Ontology Biological Process of significantly downregulated genes in RUNX3 KO cells (n = 1,005). C, Heat maps representing read densities of RUNX3, H3K27ac, H3K4me3 (promoter mark), and H3K4me1 (enhancer mark) in ±5-kb regions centered on RUNX3 binding sites. RUNX3 binding sites were classified into 642 sites around TSS and 2,492 distal to TSS (left). Enriched de novo motifs at the 642 RUNX3 binding site around TSS and the 2,492 distal regulatory regions are presented (right). D, Diagrams of H3K27ac HiChIP loops for prediction of RUNX3 target genes. Significant interactions were calculated (P E, Rate of differentially expressed genes in among the RUNX3 target genes predicted by H3K27ac HiChIP. A total of 178 and 58 genes were significantly downregulated and upregulated, respectively, by RUNX3 KO. F, Enriched pathways obtained from MSIgDB Hallmark 2020 2 and Gene Ontology Biological Process, analyzing significantly repressed RUNX3 target genes in RUNX3 KO cells.</p

Supplementary Data S1 from Aberrant Upregulation of RUNX3 Activates Developmental Genes to Drive Metastasis in Gastric Cancer

The excel file shows active RUNX3 binding sites enriched with CDX2, LHX2 and TCF3 motifs and their corresponding GO biological processes at the Pages 1, 2 and 3, respectively.</p

Supplementary Figure S3 from Aberrant Upregulation of RUNX3 Activates Developmental Genes to Drive Metastasis in Gastric Cancer

RUNX3 expression is higher in liver metastasis than in the primary tumor in splenic inoculation model A, RT-qPCR indicates relative RUNX3 expression obtained from tumors in spleen and liver metastasis after inoculation of HGC-27 control cells; **, P < 0.01 by a two-tailed Student t test. B, representative images of immunofluorescence study to compare RUNX3 expression obtained from tumors in spleen and liver metastasis after inoculation of HGC-27 control cells. Imaging for tumors of the spleen by RUNX3 KO cells is also shown as a negative control. Scale bar: 20 μm. C, quantitative analysis by image-J for fluorescence level of RUNX3 in spleen and liver metastasis after inoculation of HGC-27 control cells; **, P < 0.01 by a two-tailed Student t test.</p

FIGURE 4 from Aberrant Upregulation of RUNX3 Activates Developmental Genes to Drive Metastasis in Gastric Cancer

Histone modification and interactions proximal to genes transcriptionally activated by RUNX3. ChIP-seq analyses of histone modification and RUNX3 binding sites in HGC-27 control (cont) and RUNX3 KO cells are juxtaposed to H3K27ac HiChIP analyses. The representative genes include CD44, IGFBP3, VIM, WNT5A, DPYSL3, and RUNX2, based on the list of genes that showed high scores of P values and H3K27ac HiChIP profiles. Enhancer–promoter interactions are indicated by loops. Arrowhead indicates change in H3K27ac peak after RUNX3 KO.</p

Supplementary Figure S5 from Aberrant Upregulation of RUNX3 Activates Developmental Genes to Drive Metastasis in Gastric Cancer

Addition of exogenous recombinant WNT5A restores migration and invasion abilities to RUNX3 KO cells A, WST-1 cell proliferation assay in HGC-27 after siRNA mediated WNT5A KD; *, P < 0.05 by a two-tailed Student t test. B, immunoblot for WNT5A in HGC-27 KO cells after WNT5A recombinant treatment (0.1mg/mL) is shown. C, representative images for HGC-27 and LMSU after siWNT5A, and in GAS24 after WNT5A recombinant treatment (0.1mg/mL) in Matrigel colony formation assay (top). The number of colony formation was counted and shown as a fold change to control cells (mean + SD); **, P < 0.01 and *, P < 0.05 by a two-tailed Student t test.</p

Supplementary Figure S6 from Aberrant Upregulation of RUNX3 Activates Developmental Genes to Drive Metastasis in Gastric Cancer

Higher WNT5A expression in gastric cancer patients is associated with advanced stage TCGA gastric cancer patient dataset was visualized by XENA software (UCSC, n=591). The RNA expression level of WNT5A between stage I (n=25) and stage IV (n=41) are shown; **, P < 0.01 by a two-tailed Student t test.</p